The present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to transforming growth factor 1 (TGFxcex21). More particularly, the invention is concerned with specific binding members which include the VH CDR3 of the antibody SL15 (the antibody formerly known as Kylie), especially the SL15 VH domain, which may be in combination with the SL15A or SL15S VL domains. Furthermore, the invention relates to use of such specific binding members in pharmaceutical preparations, particularly for the treatment of fibrotic disease, the modulation of wound healing and the treatment of cancer.
PCT/GB96/02450 published as WO97/13844 discloses the isolation of human antibodies specific for human TGFxcex21 and human antibodies specific for human TGFxcex22. It describes antibodies with the 31G9 VH domain and variants of the domain. More specifically the application described the antibody CS37 that comprises the 31G9 VH domain together with the CS37 VL and variants of this domain, including antibodies which:
(i) compete in ELISA with CS37 for binding to TGFxcex21,
(ii) bind TGFxcex21 preferentially with respect to TGFxcex23, and
(iii) neutralise TGFxcex21.
The present invention is based on identification of antibodies which are related to CS37, but which have unexpectedly advantageous properties with respect to binding and neutralisation of TGFxcex21. They do not bind to, or neutralise, TGFxcex22 or TGFxcex23.
Antibodies of the present invention strongly neutralise active TGFxcex21 . The epitope for these antibodies lies in the C-terminal region of TGFxcex21, (residues 83-112) and includes the loop consisting of residues 92-98 of TGFxcex21, also known as finger 2, a region which has been identified as interacting with the receptor for TGFxcex2. The antibodies bind preferentially to active TGFxcex21 with respect to latent TGFxcex21.
Variants of SL15S that strongly neutralise TGFxcex21 are also disclosed herein. These vary mainly by amino acid substitutions in the CDR3 of VH or VL domains. There are, however, other sites where substitutions may be made, e.g. at residue 25 in the light chain an alanine may be substituted, generating the IgG4 antibody, SL15A IgG4, CAT-192. Several substitutions may be made that are compatible with the retention of strong neutralising activity. The antibodies of this invention will be particularly useful for treatment of fibrotic diseases, e.g. lung fibrosis, modulation of the scarring response, e.g. in wound healing and corneal scarring, and in other contexts discussed further below such as the treatment of tumors.
Specific binding proteins such as antibodies which are based on the complementarity-determining regions (CDRs) of the advantageous antibody VH and VL domains identified herein, particularly the CDR 3 regions, will be useful for the purposes discussed, and represent aspects of the present invention.
The most preferred embodiments of the present invention in its various aspects are based on the VH CDR3 of the SL15 VH domain identified herein, VH domains including the SL15 VH CDR3, especially the SL15 VH domain itself, and pairings of such VH domains with VL domains, especially the SL15A or SL15S VL domains, or other VL domain comprising the SL15 VL CDR3. The antibody antigen-binding domain SL15 (in whichever format, e.g. scFv or IgG4) consists of the SL15 VH and, in two variants, either the SL15A VL (CS37) or SL15S VL (CS37 with A25S). In either variant, SL15 is the antibody formerly known as Kylie. SL15S scFv is also known as CAT 191; SL15A IgG4 is also known as CAT-192, and SL15S IgG4 is also known as CAT-193.
Further embodiments of the invention in its various aspects are based on the JT182 VH CDR3, VH domains including the JT182 VH CDR3, especially the JT182 VH domain, and pairings of such VH domains with VL domains, especially the CS37 VL domain. JT182 is not as effective as SL15, but still has unexpectedly improved properties over CS37.
In a first aspect the present invention provides an isolated specific binding member capable of binding TGFxcex21, wherein said specific binding member comprises an antigen binding domain comprising a VH CDR3 with an amino acid sequence substantially as set out as VH CDR3 of SL15 or JT182 in Table 1 or Table 2.
Residues that differ between the scFv fragments are underlined.
The invention further provides said isolated specific binding member which further comprises a VH CDR1 or VH CDR2 with amino acid sequences substantially as set out as one or both of the VH CDR1 and VH CDR2 of the CS37 VH, preferably both (Table 2).
In a preferred embodiment, the binding domains are carried by a human antibody framework. One preferred example of such an embodiment is a VH domain with an amino acid sequence substantially as set out as the JT182 VH domain of which the sequence is set out in SEQ ID NO:10. A further preferred embodiment is a VH domain with an amino acid sequence substantially as set out as the SL15 VH domain, of which the sequence is set out in SEQ ID NO:4.
In a second aspect, the invention provides an isolated specific binding member capable of binding TGFxcex21, wherein said specific binding member comprises an antigen binding domain comprising a VL domain with an amino acid sequence substantially as set out as the SL15S VL domain, of which the sequence is set out in SEQ ID NO:8.
In a further aspect, the invention provides a specific binding member capable of binding TGFxcex21, comprising a VH domain as set out above with respect to the first aspect, and a VL domain, preferably wherein the VL domain has an amino acid sequence substantially as set out as the CS37 VL (SL15A), of which the sequence is set out in SEQ ID NO:6, or the SL15S VL, of which the sequence is set out in SEQ ID NO:8.
In a particularly preferred embodiment, the invention provides a specific binding member comprising the CS37 VL domain and a VH domain selected from JT182 VH and SL15 VH, most preferably SL15 VH. In a further particularly preferred embodiment, the invention provides a specific binding member comprising SL15 VH and SL15A VL (CS37 VL) or SL15S VL.
Preferred embodiments of the present invention provide specific binding members comprising the JT182 VH or SL15 VH domain in which 1, 2, 3, 4 or 5 amino acid substitutions have been made in a CDR, e.g. CDR3, and/or FR, which specific binding members retain ability to bind TGFxcex21. Further preferred embodiments provide specific binding members comprising the SL15A (CS37) VL or SL15S VL, or SL15A or SL15S VL domain in which 1, 2, 3, 4 or 5 amino acid substitutions have been made in a CDR, e.g. CDR3, and/or FR which specific binding members retain ability to bind TGFxcex21. Such amino acid substitutions are generally xe2x80x9cconservativexe2x80x9d, for instance substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. At certain positions non-conservative substitutions are allowable.
Such specific binding members are able to bind TGFxcex21. Preferred embodiments lack significant cross-reactivity with TGFxcex22 and/or TGFxcex23, preferably TGFxcex22 and TGFxcex23.
Preferred embodiments strongly neutralise TGFxcex21, having a potency of at least 5 times better than does CS37, more preferably about 10 times, 15 times, 20 times, 50 times, 75 times, 100 times or 150 times better, in a radioreceptor assay (Lucas C et al (1991)Meth in Enzymology 198, 303-16). Potency is measured with the antibody under study and CS37 in equivalent molecular formats, e.g. as monovalent antibodies (scFv or Fab) or as bivalent antibodies (IgG1 or IgG4).
Preferred embodiments bind active TGFxcex21 preferentially to latent TGFxcex21.
Variants of the VH and VL domains and CDRs of which the sequences are set out herein and which can be employed in specific binding members for TGFxcex21 can be obtained by means of methods of sequence alteration or mutation and screening. Such methods are also provided by the present invention.
In addition to antibody sequences, the specific binding member may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. Specific binding members of the invention may carry a detectable label, or may be conjugated to a toxin or enzyme (e.g. via a peptidyl bond or linker).
In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member.
Specific binding members according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention. Conditions treatable in accordance with the present invention are described below.
These and other aspects of the invention are described in further detail below.
All documents mentioned herein are incorporated by reference. Sequences described herein are shown and referred to in the conventional 5xe2x80x2 to 3xe2x80x2 and N to C terminal notation for nucleic acid and amino acids sequences respectively, unless specifically indicated otherwise.